Whole slide imaging systems have been developed that can produce a digital image of an entire microscope slide, or portions thereof. These include microscope imaging optics and digital cameras together with mechanical apparatus to sweep out a scan of the sample, along with software to coordinate these actions and store the resulting images. Typical pathology specimens can be imaged in about 10 minutes or less, and the resulting images may have a resolution of ¼ micron or finer, corresponding to 3.6 billion pixels for a 15×15 mm sample. These systems are used in the fields of research and clinical pathology to image tissue sections, cytology samples, and other samples for which microscopic imaging is valuable.
Some of these instruments produce an image based on fluorescent light emission from the sample in response to excitation light. Fluorescence imagery is useful for samples prepared with fluorescent agents such as DAPI, the Alexa fluors, the Cy fluors, and other compounds known in the art. Some such instruments use a color digital camera, together with an epi-filter cube containing a multiband excitation filter and a multiband emission filter, to generate a 3-band image of the sample, which enables measuring up to three fluors in a given scan. The Aperio ScanScope FL (available from Aperio, Vista, Calif.) is a system of this type.
Other whole-slide scanning systems produce a fluorescence image using a monochrome digital camera, together with a similar epi-filter cube and a wavelength-selective excitation source; they illuminate the sample in a first wavelength band while a first image is acquired, then illuminate it in another band while a second image is acquired, and so on. In this way, three or even four bands can be imaged, the limit being imposed by available optical filters for excitation and emission filtration. The 3DHistech P250 (available from 3DHistech, Budapest, Hungary) is an example of this kind of scanner.
Yet other fluorescence scanners use a monochrome digital camera, and engage a series of different epi-filter cubes, taking an image with each filter cube. Since each filter cube can have its own distinct excitation and emission filter, these are not limited to three or four bands. The Leica SCN400F (available from Leica Biosystems, Richmond, Ill.) is an example of a system of this kind.
Multispectral imaging systems exist that can measure more than 4 bands in a fluorescence image of a sample. The Vectra imaging system (available from PerkinElmer, Waltham, Mass.) is an example of such a system. It uses a liquid crystal tunable filter to select individual wavelengths within the visible range, under computer control, while taking images with a monochrome digital camera. The data thus acquired is termed an image cube, and it contains a spectrum at each of many spatial locations in an image. It is possible to unmix the spectrum at each point into its various component signals, using a spectral library of known spectra for the components that are present in the sample. Nuance software (available from PerkinElmer, Waltham, Mass.) can produce such spectral libraries and use them to unmix image cubes into component images.
Biological samples generally emit some fluorescent light in response to an excitation light beam, even if no exogenous fluorophores have been applied. This so-called autofluorescence signal can be significant in tissue samples, and can interfere with accurate measurement of fluorescent probe signals. The effect of autofluorescence in tissue samples can be reduced or nearly eliminated if spectral libraries used for unmixing include a signal corresponding to sample autofluorescence. If such a signal is included, the autofluorescence signal is attributed to the autofluorescence component image when an image cube for a sample with autofluorescence is unmixed, and accurate signals are obtained for the component images associated with fluorescent dyes and probes.